2'-O-Methyl-guanosine 3-base RNA fragments mediate essential natural TLR7/8 antagonism

Daily phagocytic clearance of billions of apoptotic cells is fundamental to the maintenance of homeostasis and is normally immune silent. Toll-like receptors (TLR) 7 and 8 are phagosomal innate immune sensors known to instigate inflammation in response to guanosine, uridine, and ~2-3 base long degradation products from endosomal RNAs. Exactly how TLR7 and TLR8 evade activation by degradation products from host-RNAs is currently unknown. The activity of the mouse TLR7 inhibitory sequence GGC-v1 was tested on primary bone marrow-derived macrophages (BMDMs) derived from Tlr7Y264H gain-of-function mutant mice. This Tlr7Y264H gain of function mutation results in constitutive engagement of TLR7 through an increased affinity for guanosine. Overnight treatment of Tlr7Y264H mutant BMDMs with GGC-v1 or the small molecule TLR7/8 inhibitor Enpatoran led to significant down regulation of 20 out of the 22 genes regulated by GGC-v1 that were also down-regulated by Enpatoran, indicating that TLR7 sensing was effectively dampened. Overall design: Primary bone marrow derived macrophages (BMDMs) from 9-11 week-old Tlr7Y264H heterozygous gain-of-function female mice were extracted and differentiated for 5 days in complete DMEM supplemented with L929 conditioned medium, prior to 24 h incubation with 5 µM GGC-v1 or 100 nM Enpatoran and total RNA purification for RNA sequencing.